Comparison of tryptic peptide maps of eight isoperoxidases from tobacco tissue cultures

Eight isoperoxidases from tobacco suspension culture WR-132 (termed C_n, C₃, C₄, A_c, and A_f) and tobacco callus culture W-38 (termed A₁, A₂, and A₃) have been subjected to trypsin digestion followed by peptide mapping. The peptide maps of isoperoxidases A_f and A₃ are identical. All other isoperoxidases do not appear to be dramatically dissimilar in certain portions of their sequence, since many matching peptides have been found when various isoperoxidases are cross-compared. However, only two, and possibly three, highly homologous peptides are present in all of the isoperoxidases.     

Ferulic acid: a substrate for two isoperoxidases from Nicotiana tabacum tissue cultures

An anodic isoperoxidase (A₂) from tobacco tissue culture W-38 and a cathodic isoperoxidase (C₄) from tobacco tissue suspension culture WR-132 have been separated and characterized. Both isoperoxidases catalysed oxidation of ferulic acid in the presence of H₂O₂. When the reaction mixture was subjected to TLC, ferulic acid was found to have been converted to an unknown compound which, after treatment with ammonia, fluoresces green in UV light. Both the isoperoxidases A₂ and C₄ appear to follow simple Michaelis-Menten kinetics with respect to guaiacol as the substrate. The Kms for guaiacol are 4 and 4·5 mM for isoperoxidases C₄ and A₂, respectively. The pH optimum for both enzymes is about 6·0. The effect of various phenolic and related compounds on the activity of each isoperoxidase is reported and discussed.     

Isoperoxidases from tobacco tissue cultures

Two anodic isoperoxidases (A₁ and A₂) from tobacco tissue culture W-38 and two cathodic isoperoxidases (C₃ and C₄) from tobacco suspension culture WR-132 have been separated and characterized. Molecular weights for each of the isoperoxidases have been determined by two different methods. Only C₄ contained a carbohydrate component. The substrate specificity and the pH optima for the four enzymes with each of five substrates were determined.     

Effect of darkness on isoperoxidases in tobacco tissue cultures

In order to study the effect of light on the tobacco tissue culture WR-132, 5 passages (10 days' growth per passage) of these cells were grown in darkness, and 3 passages were separately grown in intense light (16000 lx). All other growth conditions were the same. The resulting isoperoxidase patterns present in these cells and in their growth media were analyzed at 2-day intervals during this period and then compared with the isoperoxidase patterns of cells grown under dim light conditions (10 lx). A new cathodic isoperoxidase (C_n) appeared in the medium within 2 days after the cells were placed in the dark. C_n was present in all media of WR-132 cell cultures analyzed throughout the 5 passages grown in darkness. The fifth passage in darkness produced total cessation of growth (apparent death). C_n increased and new anodic isoperoxidases A_a, A_b, A_d and A_e appeared in the media as the cells approached death in darkness.     

Hydrocarbons from the essential oil of Cymbopogon martinii

The composition of the hydrocarbon fraction of the essential oil from Cymbopogon martinii, which represents less than 5% of the oil, has been studied. Using well-established techniques, 11 monoterpenes (ca 46 %), 28 sesquiterpenes (ca 52%) and 16 n-alkanes (ca 1.6%) have been identified. The major constituents are limonene, α-terpinene, myrcene, β-caryophyllene, α-humulene, β- and δ-selinenes. The study of the n-alkanes of C. martinii revealed the presence of all members of the homologous series C₁₅C₃₀.     

Molecular weights of glycollate oxidase from C3 and C4 plants determined during early stages of purification

The M_rs of glycollate oxidase (EC 1.1.3.1) (GAO) determined soon after extraction from the leaves of several C₃ and C₄ plants are reported. The enzyme isolated from the C₃ plants wheat, barley, spinach, pea and tobacco has M_r in the range 160–180 000 and is probably a homotetramer. GAO purified from pea was previously reported as a dimer and as an octamer from spinach leaves. Therefore the quaternary structure of these GAOs soon after extraction differs from that of the purified proteins. The enzymes from the C₄ plants maize and sugar cane have M_rs ca twice this value in the range 290–310 000, whilst that of the C₄ grass Panicum maximum has an M_r of 162 000. An improved spectrophotometric assay for GAO, using a non-carcinogenic dye, is described.     

Homoeolog Expression Is Modulated Differently by Different Subgenomes in <Emphasis Type="Italic">Brassica napus</Emphasis> Hybrids and Allotetraploids

Synthetic and natural allotetraploid Brassica napus (2n?=?38, AACC) have been widely used as a model to study the genetic changes associated with allopolyploidization; however, there has been little research on the homoeolog expression patterns and the roles of cis and trans regulation. Herein, homoeolog expression patterns were assessed by using RNA-seq for two interspecific hybrids (A_nC_o with the extracted A subgenome from natural B. napus, and A_rC_o with the A subgenome from extant B. rapa), synthetic and natural allopolyploids (C_oC_oA_rA_r and A_nA_nC_nC_n), and the diploid parents. The ranges of homoeolog expression bias decreased after hybridization, whereas the extents of homoeolog expression bias and non-conserved expression, especially transgressive expression, increased over evolutionary time. Despite sharing the same C subgenome parent, these two hybrids showed different homolog expression patterns in many respects. In A_nC_o, the trans-regulatory factors from C_o subgenome tended to cause downregulation of A_n subgenome homoeologs, but trans-regulatory factors from the A_n subgenome acted as both activators and repressors, and such asymmetric effects of trans-regulatory factors might explain why the homoeolog expression was biased toward the C subgenome after genome merger. No significant asymmetric effects of trans-regulatory factors were found in A_rC_o, which was consistent with the overall balanced expression of homoeologs. These results suggested that A subgenomes with different regulatory systems might act differently in modulating homoeolog expression after merger with the C subgenome, resulting in either balanced or unbalanced homoeolog expression biases.     

Epicuticular wax of Pinus radiata needles

Epicuticular wax isolated from the cotyledons and primary needles of 10-week-old Pinus radiata seedlings is similar in composition and contains 86% neutral compounds, viz. alkyl esters (25%, C₂₄–C₆₄), nonacosan-10-ol (52%), heptacosane-5,10-diol (2%), nonacosane-4,10-diol, nonacosane-5,10-diol, and nonacosane-10,13-diol (total 12%) and estolides, MW ca 800 (2%), MW ca 1100 (6%), and MW ca 1500 (1%). The acidic fraction (14%) contains n-acids (78%, C₁₂–C₃₂) and diterpene acids (22%, mainly abieta-8,11,13-trien-18-oic, with lesser amounts of pimara-8(14),15-dien-18-oic, isopimara-7,15-dien-18-oic and hydroxylated aromatic, diene and mono-ene acids). Wax isolated from primary needles of 1-yr-old seedlings had a similar neutral fraction composition, but the acidic fraction contained predominantly the diterpene acid mixture, with only trace amounts of n-acids. The wax from 1-yr-old secondary, needles from P. radiata forest trees aged 5 yr and 40 yr contained an acid fraction (12% 5 yr, 17% 40 yr trees) comprising the diterpene acid mixture, with trace amounts of n-acids together with ω-hydroxy acids (C₁₂, C₁₄ and C₁₆). The neutral fraction from both young and old trees had a similar composition containing alkyl esters (7%, C₂₄–C₆₆), estolides (90%, MW 566-ca 1500), nonacosan-10-ol (2%) and the heptacosane and nonacosane diols (1%). During growth and maturation of P. radiata, the nonacosan-10-ol content of the needle wax decreases while the proportion of estolides and diterpene acids increases, the latter probably being located around the stomatal pore.     

HORMONAL CONTROL OF PEROXIDASE ACTIVITY IN CULTURED PELARGONIUM PITH

Freshly excised Pelargonium pith tissue lacks peroxidase activity toward guaiacol or benzidine, but it develops such activity within 24–36 hr in aseptic culture. All the activity is manifested as a single enzyme moving toward the cathode during electrophoresis on starch gel at pH 9.0. This development of peroxidase activity is at first (up to ca. 50 hr in culture) inhibited and later (ca. 100–150 hr in culture) promoted by IAA. This dual effect of IAA resembles that previously reported for specific isoperoxidases in tobacco pith cells. Kinetin alone also inhibits peroxidase formation, but in the presence of IAA those concentrations which enhance growth enhance peroxidase formation as well.     

AREA AND VISUAL THRESHOLD

1. The variation of threshold with field area was measured in fields homogeneous in rod-cone composition. At 15° above the fovea, an increase in field diameter from 1° to 5° reduces the threshold sevenfold, at 25° above the fovea tenfold. 2. These changes are shown to follow qualitatively from simple statistical properties of the retinal mosaic. Analytic treatment leads to the expression, (A – n_t)^k I = C, in which A = area, n_t = constant threshold number of elements, I = threshold intensity, and k and C are constants. This equation describes the available data accurately, and is the general form of previous empirical area-threshold formulae.     

Peroxidases of Solanum melongena leaves

Three major peroxidases, designated as A, B₂ and B₂ from Solanum melongena leaves have been reported. Peroxidases-A, -B₂ and -B₂ were considered to be true peroxidases on the basis of k₁:k₄ ratio. The pH optima for the three enzymes were found to be 7·0, 6·0 and 6.0 respectively. These peroxidases differ in their k₁:k₄ ratio, in the effect of pH on this ratio and in the uric acid/guaiacol and o-dianisidine/guaiacol activity ratio.     

Enzyme Regulation in Crassulacean Acid Metabolism Photosynthesis : Studies on the Ferredoxin/Thioredoxin System of KalanchoE daigremontiana

Cell-free preparations of the Crassulacean acid metabolism (CAM) plant, Kalanchoë daigremontiana, were analyzed for thioredoxins and ferredoxin-thioredoxin reductase. Three distinct forms of thioredoxin were identified in Kalanchoë leaves, two of which specifically activated fructose 1,6-bisphosphatase (designated f₁ and f₂) and a third which activated NADP-malate dehydrogenase (thioredoxin m). The apparent molecular weight of both forms of thioredoxin f was 11,000 and that of thioredoxin m was 10,000. In parallel studies, ferredoxin and ferredoxin-thioredoxin reductase were purified from Kalanchoë leaf preparations. Kalanchoë ferredoxin-thioredoxin reductase was similar to that of C₃ and C₄ plants in molecular weight (31,000) and immunological cross-reactivity. Kalanchoë ferredoxin-thioredoxin reductase exhibited an affinity for ferredoxin as demonstrated by its binding to an immobilized ferredoxin affinity column. The purified components of the Kalanchoë ferredoxin-thioredoxin system could be recombined to function in the photoregulation of chloroplast enzymes. The data suggest that the ferredoxin/thioredoxin system plays a role in enzyme regulation of all higher plants irrespective of whether they show C₃, C₄, or CAM photosynthesis.     

小麦和玉米叶片光合-蒸腾日变化耦合机理

植物叶片光合-蒸腾耦合是陆地生态系统碳-水耦合的基础.已有研究将叶片光合-蒸腾耦合笼统归因于气孔的共同控制作用,缺乏对其耦合机理的全面分析.选择华北地区大田作物冬小麦(C3)和夏玉米(C4)为研究对象,分别在小麦开花期和玉米拔节期选择典型晴天进行叶片光合蒸腾日变化观测(8:00-18:00).结果发现:1)光合速率(An)、蒸腾速率(Tr)以及光合有效辐射(PAR)、叶片表面温度(T)和气孔导度(gs)均表现出单峰日变化特征,峰值出现在正午前后;2)An-Tr具有极显著线性正相关关系(小麦和玉米的相关系数分别为0.75**和0.92**,回归直线斜率分别为1.99和3.62);3)PAR、T和gs与An和Tr有线性正相关关系;4)PAR-An与PAR-Tr、T-An与T-Tr、gs-An与gs-Tr的回归直线形态非常相似.分析认为:1)在光合-蒸腾耦合特征方面,C3作物小麦和C4作物玉米叶片光合-蒸腾都有明显的线性耦合关系,但两者的耦合关系特征存在明显差异,玉米的An-Tr线性回归斜率要明显大于小麦;2)在光合-蒸腾耦合机理方面,日变化中PAR、T和gs同时受太阳辐射调控与An、Tr发生趋向相同、形态相似且近似同步的变化,因此PAR-An与PAR-Tr、T-An与T-Tr、gs-An与gs-Tr具有形态相似的线性关系,这保证了在PAR、T和gs等调控因子发生较大变化的日变化过程中光合-蒸腾保持良好的线性耦合关系.     

Isoperoxidase pattern and internode length genotype in Pisum

Inbred Pisum sativum lines of known constitution for the intemode length genes Le, La and Cry, and representing four height phenotypes, were grown to the 7-intemode stage in the light. Six cationic isoperoxidases, making up ca. 90% of the activity of stem extracts, were resolved by concave gradient elution from Dowex 50 columns and shown to run as single peroxidase bands on starch gel electrophoresis. They were all able to oxidise IAA in the presence of 2,4-dichlorophenol, but fell into two groups with widely differing IAA oxidase/peroxidase ratios. The isoperoxidase patterns were independent of both genotype and phenotype for internode length, thus making it unlikely that these loci exert their effect on internode extension via control of synthesis of a particular isoperoxidase. Amongst the lines screened polymorphism was detected involving two of the isoperoxidases, and limited F₂ data suggest that these two variants fire determined by alleles of a single gene. Isoperoxidase patterns of stem extracts of 6 other Pisum species did not differ significantly from the two found in P. sativum.     

Structural physiological, and biochemical gradients in tobacco pith tissue

Explants of tobacco pith taken at various distances from the apex of a mature stem show a sharp gradient in growth potential in vitro; growth is highest in the extreme apical and basal explants, and is minimal in explants removed ca. 75 cm from the apex. Calluses produced by the vigorously growing basal explants are harder and more compact than those produced from more apical explants. The gradient in growth potential is directly correlated with gradients in RNA, protein of cell sap and soluble N per unit fresh weight, but is inversely correlated with peroxidase activity. Cell size increases from apex to base of plants.

The peroxidase activity of pith explants is electrophoretically resolvable into 2 isoperoxidases, moving anodically at pH 9.0. During in vitro culture, this activity rises, due to the formation of several new isozymes moving toward the cathode. The appearance of these isozymes occurs most rapidly in apical and extreme basal explants.

     

Identification of Gibberellins A4, A9, and A24 from Phaeosphaeria sp. L487 Cultured in a Chemically Defined Medium

Gibberellins A₄, A₉, and A₂₄ were isolated and identified from the new gibberellin-producing fungus, Phaeosphaeria sp. L487 (Loculoascomycetes), cultivated in a chemically defined medium; their yields from the culture filtrate were ca. 1. 7, 0. 3, and 0.4 μg/ml, respectively. Gibberellins A₄ and A₉ significantly stimulated the hypocotyl growth of Chinese cabbage seedlings at a very low concentration of less than 0.01 μg/ml.     

Gibberellins and the Legume-Rhizobium Symbiosis : I. Endogenous Gibberellins of Lima Bean (Phaseolus lunatus L.) Stems and Nodules

The content of gibberellin-like substances in nodules formed by Bradyrhizobium species strain 127E14 on roots of lima bean (Phaseolus lunatus L.) has been previously found to be relatively high. The objectives of the present study were to purify and identify the endogenous gibberellins from the stems and nodules of lima bean. By sequential silica gel partition column chromatography, C₁₈ reverse-phase high performance liquid chromatography, and combined gas chromatography-mass spectrometry, the gibberellins A₁, A₃, A₁₉, A₂₀, A₂₉, and A₄₄ were identified from root nodules. Gibberellins A₁, A₃, A₁₉, A₂₀, and A₄₄ were also identified from lima bean stem tissue. These data provide the first mass spectral-based evidence that gibberellins are present in leguminous root nodules. The presence of the gibberellins identified indicates that the early 13-hydroxylation gibberellin biosynthetic pathway predominates in stem and nodule tissue. However, it is not known if the gibberellins within the nodules are produced in situ, or if they are imported from some remote host plant tissue.     

Isolation and some properties of extracellular d-glucosyltransferases and d-fructosyltransferases from Streptococcus mutans serotypes c, e, and f

Extracellular d-glucosyltransferases (GTase) and d-fructosyltransferases (FTase) were isolated from Streptococcus mutans IB (serotype c), B14 (e), and OMZ175 (f) by chromatofocusing, followed by hydroxyapatite column chromatography. The GTases isolated from serotypes c, e, and f are basic proteins (pI 7.4). The serotype c and e enzymes have two protein components having M_r 173 000 and 158 000 and the enzyme of the serotype f one component having M_r 156 000. The GTases of all the serotypes showed a K_m value for sucrose of 10–14mm and an optimum pH 5.5–6.0 for enzyme activity, and their activities were enhanced by the presence of primer Dextran T10. The α-d-glucans synthesized by the purified GTases are water soluble and primarily consist of (1→6)-α-d-glucosidic linkage (41–66 mol/100 mol) and α-d-(1→3,6)-branch linkage (6–20 mol/100 mol), but significant proportions of α-d-(1→3), α-d-(1→4), and α-d-(1→3,4) linkages (11, 6, and 14 mol/100 mol, respectively) were detected in the serotype c α-d-glucan. The isolated FTases of the serotypes c, e, and f are acidic enzymes (pI 4.6) and consist of two components having M_r 84 000 and 76 000 for the serotype c enzyme, and 106 000 and 84 000 for the serotypes e and f enzymes, respectively. The K_m value for sucrose was 6, 10, and 17mm for the serotypes c, e, and f enzymes, respectively, and the optimum pH of enzymic activity 5.5–6.0. Reactivity with Concanavalin A, susceptibility to acid hydrolysis, and paper chromatography of the hydrolyzates suggested that the water-soluble β-d-fructans synthesized by the purified FTases were of the inulin-type and had chemical structures somewhat different among the serotypes.     

Ribonuclease activities in bean roots

Vicia faba meristematic and elongating root cells (zones 0–4 and 10–20 mm) contained one nuclease (A₁) and four ribonucleases (A₂, A₃, C₁, C₂). When the overall activity of each enzyme was expressed per cell, the elongating cells contained 4-, 4-, 4-, 10- and 17-fold more activity than meristematic cells for A₁, C₁, C₂, A₂ and A₃, respectively.     

Aerosol-assisted chemical vapour deposition (AACVD) of silver films from triorganophosphine adducts of silver carboxylates, including the structure of [Ag(O2CC3F7)(PPh3)2]

Silver carboxylates [Ag(O₂CR): R=Me, ^tBu, 2,4,6-Me₃C₆H₂], fluorocarboxlyates [Ag(O₂CR_f): R_f=C₃F₇, C₆F₁₃, C₇F₁₅] and their phosphine adducts [Ag(O₂CR)·nPR₃′: R=Me, ^tBu, 2,4,6-Me₃C₆H₂, R′=Me, Ph, n=2; R=Me, R′=Me, n=3; Ag(O₂CR_f).2PPh₃, R_f=C₃F₇, C₆F₁₃, C₇F₁₅] have been synthesised, characterised spectroscopically and used as precursors in the aerosol-assisted chemical vapour deposition of silver films. All the phosphine adducts produced films, though in general PMe₃ adducts, proved more successful than PPh₃ analogues. The fluoro-carboxylates and their PPh₃ adducts all generated silver films, though the growth rate for the adducts was lower. All these latter films showed carbon impurities while fluorine was also evident in most cases. The X-ray structure of AgO₂CC₃F₇·2PPh₃ is also reported.